The present invention is in the technical field of biomedical imaging, more particularly in the technical field of optical microscopy and more particularly in the technical field of optical sectioning microscopy.
Optical sectioning microscopy refers to the imaging of a section in depth of a sample, the section being in the focal plane of a microscope objective, while removing the background in the image due to out-of-focus light and scatter. When microscopy is applied to biological samples such as cells or tissues which exhibit strong scattering, optical sectioning dramatically improves the quality of the images obtained. It is thus of paramount importance for biological as well as medical purposes. Among the techniques that are used for high resolution imaging in the micrometer range, confocal microscopy (see for example U.S. Pat. No. 3,013,467—Minsky) and non-linear microcopies (e.g. two Photon—see for example Denk W, Strickler J, Webb W (1990). “Two-photon laser scanning fluorescence microscopy”. Science 248 (4951): 73-6) are now widely used. They take advantage of large numerical aperture microscope (NA˜0.8 to 1.4) objectives to achieve the required limited depth of field that insures good sectioning ability.
Optical Coherence Tomography (OCT) is an efficient optical sectioning technique for in-depth imaging of biological tissues. OCT relies on interferometric selection of ballistic photons (see for example J. G. Fujimoto et al., Optical biopsy and imaging using optical coherence tomography, Nature Med. 1, 970-972 (1995)). It has proved to be a highly valuable tool for biomedical imaging in particular in the field of eye examination. Concerning the other tissues of the human body that are dense and exhibit a very large scattering cross-section it is more difficult to get sharp images at the cellular level of virtual thin slices at large depths.
In contrast with most of the available OCT approaches (e.g. time domain OCT or Fourier domain OCT) Full Field OCT (FF-OCT) (see for example A. Dubois et al., High-resolution full-field optical coherence tomography with a Linnik microscope, Appl. Opt. 41, 805-812 (2002)) directly takes “en face” high resolution images (typically 1 μm, isotropic) using megapixels cameras coupled to gel or water immersion microscope objectives for a better matching of the refractive index of tissues.
The principle of a full-field OCT system is represented on FIG. 1. It relies on the use of thermal sources or arcs or LEDs that are spatially and temporally incoherent, coupled to an imaging interferometer, for example in the Linnik configuration as shown on FIG. 1. The FF-OCT system 100 comprises a source of partially coherent light 101, e.g. an halogen light source, optically integrated in a Köhler illuminator so as to provide uniform illumination in the sample, a beamsplitter 102, e.g. a non-polarizing beamsplitter cube, defining two interferometric arms. For a Linnik configuration, both arms include a microscope objective of the same characteristics 103 and 104. In one arm, further named the reference arm, a uniform reflective surface 105 is positioned at the focal plane of the objective and linked to an oscillator 111, allowing modulation of the optical path length of the reference arm, e.g. a piezo electric transducer. In the other arm, further called object arm, a volume and scattering sample 106—to be imaged—is positioned at the focal plane of the objective 103. An adjustable dispersion balance system is included in both arms, e.g. rotating glass plates 109 and 110. A tube lens 107 is placed at the output of the interferometer in order to conjugate the focal planes of both objectives 103 and 104 with a multichannel detector 108, e.g. a CCD or CMOS matrix. The magnification of the imaging interferometer is fixed, defined by the focal characteristics of the microscope objective 103 and tube lens 107. The adjustment in depth of the device 100 relative to the sample 106 is performed using translation means, e.g. a motorized linear translation stage.
Due to the broad spectrum of the source, interferences occur only when the optical path lengths of the two arms of the interferometer are identical within typically 1 μm. Moreover the spatial incoherence of the source prevents cross talk between pixels. The signal is extracted from the background of incoherent backscattered light using a phase-shifting method. Due to the randomness of the tissues structures, it is possible to record only two interferometric images, using a CCD or a CMOS camera for example, the phase being changed by π in the interferometer reference channel between each image and to compute the tomographic image by calculating the normalized difference of the interferometric images.
The FF-OCT system—or at least part of its components—can be displaced to move the focal plane at different depths within the sample in order to obtain 2D images at different depths that can be further reconstructed into 3D images, which we will call 3D tomographic images. En face capture allows the FF-OCT system to operate with high lateral resolution (typically ˜1 μm) using medium aperture microscope objectives (for instance, but not limited to 10× or 20× water immersion objectives with 0.3 to 0.5 numerical aperture). In addition, micrometric displacement of the FF-OCT system will enable 3D tomographic images at a 1 micron or less vertical resolution.
FF-OCT allows for ultra high resolution (typically ˜1 μm in 3D) images of scattering samples such as biological tissues, in depth, using a non-invasive process. Typical morphologic structures of tissue show sizes starting from a few microns to a few tens of microns. For example the size of a cell nucleus is typically between 5 and 10 μm, the size of a typical adipose cell between 25 and 50 μm. Due to its high resolution capabilities, the FF-OCT technique is able to provide cellular resolution images of morphologic characteristics of tissues, compared to conventional OCT techniques such as Time-Domain OCT or Fourier-Domain OCT, which have a limited transverse resolution of about 10 μm, preventing from resolving fine cellular structures. It has been proven that the use of the FF-OCT on various tissues is of great interest for pathologists (see “Jain M, Shukla N, Manzoor M, Nadolny S, Mukherjee S. Modified full-field optical coherence tomography: A novel tool for rapid histology of tissues. J Pathol Inform 2011; 2:28”), as a new tool for fast, non-invasive, non-sacrificial assessment method on freshly excised tissue. Applications such as biobanking, embryology, or surgery guidance have been raised by pathologists. For example FF-OCT might be used in the future for evaluation of surgical margins, or as a confirmation tool for assessing the adequacy of biopsied tissue for subsequent definitive histopathological diagnoses. The main advantage is that, for some clinical situations, the technique can provide significant pathological and architectural information within minutes, without the need to prepare histology slides, which a time-consuming, operator-dependant process.
However, compared to histology slides, which are the “Gold Standard” in pathology, the information given by standard FF-OCT images is mainly limited to morphology and architecture. Indeed, the contrast provided by FF-OCT results from the local variation of the amount of ballistic photons due to local refractive index variations linked to tissue structures. The contrast risen by the FF-OCT technique on particular metabolic structures such as tumorous areas of a tissue, is not always enough to perform an accurate diagnostic. In standard histology, pathological areas are often revealed using chemical coloration, such as H&E staining (Hematoxylin and Eosin). After coloration and histology slide preparation, cancerous tissue usually appears in a different color compared to the surrounding healthy tissue, with sharp contrast based on coloration. Moreover, such a contrast can be used at high magnification on a microscope to observe fine cellular structures such as cell nuclei. The size and density of nuclei is often considered as a good indicator of cancerous invasion, the size of the nuclei of cancerous cell being larger than the nuclei of healthy cells. As a consequence, when using FF-OCT microscopy on biological tissues, it may be useful for pathologists to enhance the contrast of specific cellular structures, such as cell nuclei or cancerous tissue areas. Such an additional contrast can provide both morphologic and metabolic information in a single image, approaching the information given by standard histology slides.
In standard microscopy, this additional contrast can be obtained using coloration fluids in histopathology, or fluorescent agents such as GFP (Green Fluorescent Protein) in biology. For example fluorescence spectroscopy has been used to detect early dysplasia in various organs (see Ramanujam N., Fluorescence spectroscopy of neoplastic and non-neoplastic tissues, Neoplasia 2 (1-2), 89-117 (2000)). The principle of fluorescence microscopy relies on the addition of a fluorescent agent to the sample—sometimes with specificity to a cellular structure—followed by the illumination of the sample using light with a spectrum corresponding to the absorption spectrum of said fluorescent agent. The subsequent fluorescence emission is characterized by a shifted spectrum compared to the absorption spectrum, usually towards higher wavelengths.
In conventional fluorescence microscopy of biological objects, the illumination light passes through a volume of the sample, so that the emission of fluorescence also occurs at out of focus planes. The resulting image is then blurred by a fluorescent background noise. As a consequence, fluorescence microscopy is usually used in combination with an optical sectioning technique, which allows for collecting light only coming from the sectioning plane, thus removing out of focus fluorescent noise.
However, OCT techniques, including the FF-OCT technique, are not compatible with fluorescence microscopy as light coming from the emission of fluorescence of the sample is incoherent with the light source. The properties of fluorescent photons are not correlated to the properties of the low-coherence source used for coherence tomography. Consequently, the optical path length equalization between the two interferometric arms is not possible anymore, and interference signals necessary to coherence tomography techniques can not be obtained.
One objective of the present invention is to provide additional contrast(s) in an FF-OCT system.